The development of the fertilized zygote into a complex organism has traditionally thought to be a unidirectional process, with cells in the developing fetus becoming gradually more committed to a specific tissue type. The recent development of mammalian cloning by nuclear transfer (NT) suggests that the mammalian oocyte has the remarkable ability to relieve the constraints imposed by cellular differentiation and return an adult nucleus to a totipotent, embryonic state. Thus, cloning by NT provides a unique opportunity to elucidate the molecular and cellular mechanisms by which an adult cell can be returned to an undifferentiated state, a process termed developmental reprogramming. Aim 1) To determine whether mice can be cloned directly from terminally differentiated cells. Cloned mice have only been generated from terminally differentiated cells using an embryonic stem (ES) cell intermediate, suggesting that passage of the nucleus through an ES cell might be necessary for complete reprogramming. Experiments will be carried out to determine whether this is true or if instead, the oocyte and embryo alone can successfully reprogram the epigenetic state of a terminally differentiated nucleus. Aim 2) To investigate if the transition from a differentiated state to a pluripotent state can be understood through the genome-wide changes in transcriptional activity taking place after NT. Towards this end, the genome wide changes in transcriptional activity taking place after NT are being assessed by microarray analysis. Experiments to characterize the functional importance of these observed gene expression changes between fertilized and cloned preimplantation embryos will be carried out. The experiments proposed here combine molecular, genetic and developmental approaches that will test the limits of reprogramming, elucidate mechanisms governing reprogramming and determine how inadequacies in reprogramming may lead to the inefficient nature of cloning. These studies may have profound importance for evaluating the medical utility of NT technology, deciphering the molecular basis of pluripotency and expanding our understanding of embryonic development and stem cell biology.